JP2002542784A - Gluconobacter suboxydans sorbitol dehydrogenase, its genes and methods of use - Google Patents

Abstract

  (57) [Summary] The present invention relates to the fields of molecular biology, bacteriology and industrial fermentation. More specifically, the present invention relates to an isolated nucleic acid molecule encoding the three subunits of the novel Gluconobacter oxydans sorbitol dehydrogenase (SDH) bound to a membrane of the present invention, and a vector comprising the isolated nucleic acid molecule. And a host cell. The present invention further provides isolated polypeptides for the three subunits of the SDH enzymes of the present invention, and processes for the production of L-sorbose and 2-keto-L-gulonic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to the fields of molecular biology, bacteriology and industrial fermentation. More particularly, the present invention relates to the identification and isolation of nucleic acid sequences and proteins for novel membrane-bound sorbitol dehydrogenase subunits in Gluconobacter suboxydans. The invention further relates to the fermentative production of L-sorbose from D-sorbitol and the subsequent production of 2-keto-L-gulonic acid.

2. Related Art Sorbitol dehydrogenase (SDH) is an important precursor for vitamin C synthesis in the process of producing 2-keto-L-gulonic acid (2-KLG). It is an enzyme responsible for the efficient conversion of D-sorbitol to sorbose. Gluconobacter has several SDHs,
These SDHs can be classified according to their cofactor requirements: (1) NAD-dependent, (2) NADP-dependent and (3) NAD (P) -independent. Among these, NAD (P) -independent enzymes are believed to be directly involved in the efficient production of sorbose during industrial sorbose fermentation (Cummins, JT et al., J. Bio).
l. Chem. 224, 323; 226, 301 (1957)).

[0003] Processes for producing L-sorbose from D-sorbitol typically involve acetic acid bacteria (eg, Gluconobacter suboxydans and Acet).
(X. xylinum). At room temperature,
96% -99% conversion takes place in less than 24 hours (Liebster, J. et al.
Chem. List. , 50: 395 (1956)).

[0004] L-sorbose produced by the action of SDH is a substrate in the production of 2-keto-L-gulonic acid (2-KLG). Various processes for the production of 2KLG are known. For example, the fermentative production of 2-KLG via oxidation of L-sorbose to 2-KLG via a sorbosone intermediate has been described for a process that utilizes a wide variety of bacteria: Gluconoba.
cter oxydans (U.S. Pat. Nos. 4,935,359; 4,960)
No. 5,692; No. 5,312,741; and No. 5,541,108);
Pseudogluconobacter saccharoketogene
s (US Pat. No. 4,877,735; EP 221 707); Pse
udomonas sorbosoxidans (U.S. Pat. No. 4,933,28)
Nos. 9 and 4,892,823); microorganisms from these and other genera (eg, Acetobacter, Bacillus, Serratia, Myc)
obacterium and Streptomyces (U.S. Pat. No. 3,912)
No. 3,907,639; and 3,234,105); and novel bacterial strains (US Pat. No. 5,834,231).

[0005] A number of enzymes involved in the fermentative oxidation of sorbitol, sorbose and sorbosone have been identified in the literature. U.S. Patent No. 5,88,786;
Nos. 861,292; 5,834,263 and 5,753,481 disclose nucleic acid molecules encoding L-sorbose dehydrogenase and L-sorbosone dehydrogenase and / or L-sorbose dehydrogenase and L-sorbose dehydrogenase.
-Discloses an isolated protein for sorbosone dehydrogenase; and U.S. Patent No. 5,747,301 discloses an enzyme having specificity for sorbitol dehydrogenase.

[0006] In addition to distinguishing Gluconobacter SDH based on cofactor requirements, other physical features that distinguish these different enzymes can be found in the literature. For example, sorbitol dehydrogenase identified in US Pat. No. 5,747,301 has a subcellular location (membrane-bound) and a haloenzyme molecular weight of 800 ± 50 kDa (10 homologous subunits of 79 ± 5 kDa). Are distinguished based on Shinagawa et al. (E. Shinagawa, K. Mats)
usita, O.M. Adachi and M.A. Ameyama (Agric. Bi
ol. Chem. , 46, 135-141, 1982)), the membrane-bound D-sorbitol dehydrogenase is 63,000, 51,000.
And three subunits with a molecular weight of 17,000.

[0007] In an effort to improve the productivity of commercial fermentations in the production of 2KLG, the inventors have determined that G. cerevisiae differs from other strains described in the literature. A novel sorbitol dehydrogenase bound to a membrane in a strain of suboxydans was identified (Choi, ES et al., FEMS Microbiol. Lett.
, 125: 45 (1995)).

SUMMARY OF THE INVENTION [0008] The present invention relates to a novel sorbitol dehydrogenase bound to the membrane of Gluconobacter suboxydans. The isolated sorbitol dehydrogenase enzyme contains three subunits: pyrroloquinoline quinone (py
rroloquinoline quinone (PQQ) as a cofactor; a 75 kDa first subunit that contains cytochrome c; a 50 kDa second subunit that is a cytochrome c; 3 subunits.

[0009] The present invention provides nucleic acid molecules for the three protein subunits of Gluconobacter sorbitol dehydrogenase described herein. In a first particular embodiment, the present invention provides a method comprising the steps of:
Provided for an isolated nucleic acid molecule for the SDH subunit (75 kDA).
In a second particular embodiment, the present invention provides a method for treating a second S identified by SEQ ID NO: 2.
Provided is an isolated nucleic acid molecule for a DH subunit (50 kDA). Third
In certain embodiments of the invention, the invention relates to a third SDH identified by SEQ ID NO: 3.
Provided is an isolated nucleic acid molecule for a subunit (29 kDA). Other related embodiments relate to vectors, processes for producing the same, and host cells carrying the vectors.

[0010] The invention also provides an isolated nucleic acid molecule encoding the three subunits of SDH of the invention. In one particular embodiment, the invention provides a cloned nucleic acid molecule encoding a 75 kDa subunit and a 50 kDa subunit. The structural genes for the first and second subunits of sorbitol dehydrogenase were 2,265 bp and 1
5.7 kb Pst1 DNA, 437 bp and defining the operon
It forms clusters in cloned nucleic acid molecules that are fragments.
In another specific embodiment, the invention provides a cloned nucleic acid molecule encoding a third 29 kDa SDH subunit protein. The structural gene encoding the third subunit is 921 bp in size and has a 4.5 kb C
found in the laI DNA fragment. Other related embodiments relate to vectors, processes for making the same, and host cells containing the vectors.

[0011] The present invention also relates to isolated polypeptides for the three subunits of SDH described herein.

[0012] The present invention also provides a method for the production of D-sorbose, the method comprising:
a) a polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 1; SEQ ID NO: 2
Transforming a host cell with at least one isolated nucleotide sequence selected from the group consisting of a polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 3; and (b)
Selecting and growing the transformed host cell.

Another aspect of the present invention relates to a method for the production of 2-KLG, comprising:
a) a polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 1; a polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 2; and at least one isolation selected from the group consisting of a polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 3. Transforming a host cell with the transformed nucleotide sequence; and (b) selecting and growing the transformed host cell.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 1. Definitions Cloning vector: a plasmid or phage DNA or other DNA sequence capable of autonomous replication in a host cell, and wherein such a DNA sequence is a vector Characterized by one or a few restriction endonuclease recognition sites, which can be cut in a determinable manner without losing the essential biological functions of the DNA fragment, and into which a DNA fragment can be spliced resulting in its replication and cloning. Plasmid or phage DNA or other DNA to be attached
Array. The cloning vector can further include a marker suitable for use in identifying cells transformed with the cloning vector. For example, the marker provides for tetracycline resistance or ampicillin resistance.

Expression: Expression is the process by which a polypeptide is produced from a structural gene. This process involves transcription of the gene into mRNA and such mRNA into a polypeptide.
Including translation of RNA.

Expression vector: A vector that is similar to a cloning vector, but that can enhance expression of a gene cloned into the vector after transformation into a host. The cloned gene is usually placed under the control of (ie, operably linked to) certain regulatory sequences (eg, promoter sequences). Promoter sequences can be either constitutive or inducible.

Gene: D containing information necessary to express a polypeptide or protein
NA sequence.

Host: Any prokaryotic or eukaryotic cell that is the recipient of a replicable expression or cloning vector. As used herein, "host" also includes prokaryotic or eukaryotic cells, which can be genetically engineered by well known techniques to include the desired gene in its chromosome or genome. . For examples of such hosts, see Sambrook et al., Molecula.
r Cloning: A Laboratory Manual, 2nd edition, Co
ld Spring Harbor Laboratory, Cold Spr
ing Harbor, N.M. Y. (1989).

Homologous / heterologous: The nucleotide sequence of two nucleic acid molecules is determined by the HASH encoding algorithm (Wilber, WJ and Lipman, DJ, Proc. Na
tl. Acad. Sci. 80: 726-730 (1983)), the two nucleic acid molecules are "homologous" if they share more than 50% similarity.
Is assumed to be Two nucleic acid molecules are considered "heterologous" if the nucleotide sequences of the two nucleic acid molecules share less than 50% similarity.

Mutation: As used herein, the term refers to a single base pair change, insertion or deletion in the nucleotide sequence of interest.

Mutagenesis: As used herein, the term refers to the process of producing mutations in DNA. When using “random” mutagenesis, the exact mutation site is unpredictable, occurs anywhere on the chromosome of the microorganism, and results in the mutation as a result of physical damage caused by factors (eg, radiation or chemical treatment). It is.

Operon: As used herein, the term refers to a unit of bacterial gene expression and regulation, including structural genes and regulatory elements in DNA.

Parent strain: As used herein, this term refers to a microorganism strain that has been subjected to some form of mutagenesis to give rise to a microorganism of the invention.

[0024] Phenotype: As used herein, the term refers to observable physical characteristics that depend on the genetic makeup of the microorganism.

Promoter: A DNA sequence located close to the start codon, commonly described as the 5 'region of a gene. Transcription of the adjacent gene is initiated at the promoter region. If the promoter is an inducible promoter, the rate of transcription is increased in response to the inducing factor. In contrast, when the promoter is a constitutive promoter, the rate of transcription is not regulated by the inducing factor.

Recombinant host: In accordance with the present invention, a recombinant host can be any prokaryotic or eukaryotic cell that contains the desired cloned gene on an expression or cloning vector. The term is also meant to include prokaryotic or eukaryotic cells that have been genetically engineered to contain the desired gene in the chromosome or genome of the organism.

Recombinant vector: Any cloning vector or expression vector containing the desired cloned gene.

2. Isolation and Purification of Sorbitol Dehydrogenase The present invention uses a series of column chromatography steps to transform G. sorbitol dehydrogenase. suboxyd
and KCTC (Korea Culture Type Collection)
on (Genebank) 2111 (equivalent to ATCC 621) is isolated and purified from the plasma membrane. The biochemical properties of the purified enzyme are provided, as well as the isolation of each subunit and an amino acid sequence analyzer (Applied Bis).
The determination of the N-terminal amino acid sequence of each subunit using ossystems, 477A) is provided.

[0029] The newly characterized enzymes are described in suboxydans IFO 3254
Strains (Shinagawa, E. et al., Agric. Biol. Chem., 46:
135 (1982)), contains pyrroloquinoline quinone (PQQ) as a cofactor and contains three subunits (Choi, ES, et al., FEMS Microbiol. Lett.). ., 12
5:45 (1995)).

[0030] The SDH of the present invention can be isolated using standard protein techniques. In short, G. suboxydans KCTC 2111 is a SYP medium (5%
Cultured in D-sorbitol, 1% Bacto-Peptone, 0.5% yeast extract) and the cells are buffered in 10 mM sodium acetate buffer (pH 5).
. Dissolved in 0). After centrifugation at 12,000 g to remove cell debris,
The supernatant is centrifuged in an ultracentrifuge to collect the cytoplasmic membrane fraction. Purification was performed by purifying the cytoplasmic membrane fraction with 1.5% n-octylglucoside (Boehringer).
Mannheim) and a series of chromatography columns (CM-TSK 650 (S) (Merck), DEAE-TSK 650 (S
) (Merck), Mono-S (Pharmacia) and Superos
e 12 (including Pharmacia).

The purified enzyme is a polyol (eg, D-sorbitol (100%), D-sorbitol
-Active against mannitol (68%) and D-ribitol (70%)). The activity of this enzyme increases up to 9-fold when pyrroloquinoline quinone (PQQ) is added. This suggests that PQQ is a cofactor for this enzyme; fluorescence spectroscopy confirmed that the purified enzyme contained pyrroloquinoline quinone (PQQ). The absorption spectrum analysis of the purified enzyme
It demonstrates that this enzyme contains cytochrome c. When the purified enzyme was subjected to polyacrylamide gel electrophoresis (PAGE) at pH 4.3 (Reisfeld)
, R .; A. Nature, 195: 281 (1962)), the purified enzyme forms a single active band after activity staining on this gel.

The first SDS-PAGE analysis of the purified enzyme showed that the enzyme was 75 kDa, 5
It was shown to be composed of three subunits of 0 kDa and 14 kDa.
These subunits were respectively named first, second and third subunits (Choi, ES et al., FEMS Mi).
crobiol. Lett. , 125: 45 (1995)). However, further studies of this enzyme found that another 29 kDa subunit plays a very important role in the stability and catalytic activity of SDH. That is, DE
While investigating various pH conditions in an effort to increase protein separation capacity during purification on an AE-TSK column, a partially separated activity peak eluting at pH 5.0 was detected at pH 6.0. Was found to completely separate into two separate activity peaks when eluted with. The first eluting activity peak rapidly lost enzyme activity, and the later eluting activity peak remained stable. By SDS-PAGE analysis of these two peaks, a peak having stable enzyme activity was obtained.
It was found that the peak containing the additional subunit of 29 kDa in addition to the 75 kDa and 50 kDa subunits, while the peak that rapidly lost enzymatic activity contained only the 75 kDa and 50 kDa subunits. This 2
The additional 9 kDa subunit was renamed the third subunit: whether the 14 kDa subunit previously assigned to the third subunit was the true subunit of this enzyme, the relative amount was determined by acrylamide gel. Suspicious when compared to other subunits. Further raising the pH of the elution buffer to pH 6.5
It has also been found to result in complete separation of the three subunits into individual subunits.

[0033] Different combinations of two or three subunits were identified as Ferric-Dupan
ol assay (Wood, WA, et al., Meth. Enzymol, 5:28).
7 (1962)), when tested for recovery of enzyme activity,
It was found that it was fully restored only in the presence of the 29 kDa third subunit. Therefore, it was concluded that the 29 kDa third subunit plays an important role in SDH stability and catalytic activity.

When D-sorbitol was used as a substrate, Michaelis-Mente
The n constant was determined to be Km = 120 mM and Vmax = 3.9 × 10 ⁻⁵ M / min. Dichlorophenol iodophenol (DCIP) or ferricyanide effectively acted as an electron acceptor for this enzyme. When phenazine methosulfate (PMS) was added as an electron mediator, the enzyme activity increased. The addition of calcium or magnesium ions significantly increased the activity of the purified enzyme, while the addition of copper ions severely inhibited the activity.

Further details of the purification and characterization of the SDH enzymes of the present invention are provided in Example 1.

3. Nucleic Acid Molecules of the Invention The present invention provides isolated nucleic acid molecules that encode one or more of the three subunits of the SDH enzymes described herein. Methods and techniques designed for the manipulation of isolated nucleic acid molecules are well-known in the art. For example, methods and techniques for the isolation, purification and cloning of nucleic acid molecules, and the use of eukaryotic and prokaryotic host cells and the expression of nucleic acids and proteins therein, are described in Sambrook et al. Molecular Clo
ning: A Laboratory Manual, 2nd edition, Cold Sp
ring Harbor, N.M. Y. , 1989, and Current Pro
tocols in Molecular Biology, Frederic
kM. Ausubel et al., Ed., John Wiley & Sons, Inc.
, 1987, the disclosures of which are incorporated herein by reference.

More specifically, the present invention provides a number of isolated nucleic acid molecules encoding each of the 75 kDa, 50 kDa and 29 kDa subunit proteins of the SDH enzymes of the present invention. further,
The present invention provides a number of isolated nucleic acid molecules that encode one or more of the subunit proteins of the SDH enzymes of the present invention. For clarity, certain isolated nuclear molecules of the invention are described. Thereafter, the specific properties and characteristics of these isolated nucleic acid molecules are described in more detail.

Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein are converted to an automated DNA sequencer (eg,
Applied Biosystems, Inc. Model 373A)
And the entire amino acid sequence of the polypeptide encoded by the DNA molecule determined herein was deduced by translation of the DNA sequence determined above. Therefore, any DNA determined by this automated approach
As is known in the art for sequences, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical to the actual nucleotide sequence of the sequenced DNA molecule, more typically at least about 95% to at least about 99.9% identical. . The actual sequence may be more accurately determined by other approaches, including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence, as compared to the actual sequence, causes a frameshift in the translation of the nucleotide sequence, and as a result, The deduced amino acid sequence encoded by the resulting nucleotide sequence is completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, starting from such an insertion or deletion.

[0039] In one embodiment, the present invention provides a first (of a SDH enzyme of the invention comprising:
75 kDa) providing an isolated nucleic acid molecule for a subunit: (a) a polynucleotide of SEQ ID NO: 1; (b) a polynucleotide fragment of at least about 20 nucleotides in length of the polynucleotide of SEQ ID NO: 1; A polynucleotide encoding the amino acid sequence of SEQ ID NO: 4; and (d) a polynucleotide encoding a fragment of at least about 10 amino acids in length of the amino acid sequence of SEQ ID NO: 4.

In another embodiment, the present invention provides a polyhedral polymorphism at least about 95% identical to the isolated nucleic acid sequence for the first (75 kDa) subunit of the SDH enzyme of the present invention described above. The first (75k) of the SDH enzyme of the invention comprises nucleotides.
Da) An isolated nucleic acid molecule for the subunit is provided.

Another embodiment of the present invention provides an isolated nucleic acid molecule for the second (50 kDa) subunit of the SDH enzyme of the present invention, comprising a polynucleotide sequence selected from the group consisting of: (B) a polynucleotide fragment of at least about 20 nucleotides in length of the polynucleotide of SEQ ID NO: 2; (c) a polynucleotide encoding the amino acid sequence of SEQ ID NO: 5; And (d) a polynucleotide encoding a fragment of at least about 10 amino acids in length of the amino acid sequence of SEQ ID NO: 5.

In another embodiment, the present invention provides a polyhedral polymorphism at least about 95% identical to the isolated nucleic acid sequence for the second (50 kDa) subunit of the SDH enzyme of the present invention described above. Provided is an isolated nucleic acid molecule for a second (50 kDa) subunit for an SDH enzyme of the invention, comprising a nucleotide.

Another embodiment of the present invention provides an isolated nucleic acid molecule for the third (29 kDa) subunit of the SDH enzyme of the present invention, comprising a polynucleotide sequence selected from the group consisting of: (A) the polynucleotide of SEQ ID NO: 3; (b)
A polynucleotide fragment of at least about 20 nucleotides in length of the polynucleotide of SEQ ID NO: 3; (c) a polynucleotide encoding the amino acid sequence of SEQ ID NO: 6; and (d) a fragment of at least about 10 amino acids in length of the amino acid sequence of SEQ ID NO: 6. A polynucleotide encoding

In another embodiment, the present invention provides a polyhedral polymorphism at least about 95% identical to the isolated nucleic acid sequence for the third (29 kDa) subunit of the SDH enzyme of the present invention described above. Provided is an isolated nucleic acid molecule for a third (29 kDa) subunit for an SDH enzyme of the invention, comprising a nucleotide.

Another embodiment of the present invention provides a first (75 kDa) subunit protein and a second (50 kDa) subunit of the SDH enzyme of the present invention comprising a polynucleotide sequence selected from the group consisting of: Providing isolated nucleic acid molecules encoding both proteins: (a) the polynucleotide of SEQ ID NO: 7; and (b)
A polynucleotide fragment of at least about 20 nucleotides in length of the polynucleotide of SEQ ID NO: 7.

In another embodiment, the invention relates to a nucleic acid molecule comprising at least one of the first (75 kDa) subunit protein and the second (50 kDa) subunit protein of the SDH enzyme of the invention. Provided is an isolated nucleic acid molecule for a first (75 kDa) subunit protein and a second (50 kDa) subunit protein of an SDH enzyme of the invention, comprising a polynucleotide that is about 95% identical.

Another embodiment of the present invention provides an isolated nucleic acid molecule for the third (29 kDa) subunit of the SDH enzyme of the present invention, comprising a polynucleotide sequence selected from the group consisting of: (A) a polynucleotide of SEQ ID NO: 8; and (b) a polynucleotide fragment of at least about 20 nucleotides in length of the polynucleotide of SEQ ID NO: 8.

[0048] In another embodiment, the present invention relates to a single nucleic acid molecule that is at least about 95% identical to the isolated nucleic acid molecule for the third (29 kDa) subunit of the SDH enzyme of the present invention described above. A third aspect of the SDH enzyme of the invention, comprising an isolated nucleic acid molecule.
An isolated nucleic acid molecule for the (29 kDa) subunit is provided.

By “isolated” nucleic acid molecule is intended a nucleic acid molecule, DNA or RNA, removed from its native environment. For example, a recombinant DNA molecule contained in a vector is considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA maintained in a heterologous host cell.
Molecules or (partially or substantially) purified DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of a DNA molecule of the present invention. Isolated nucleic acid molecules according to the present invention further include those produced synthetically.

[0050] RNA vectors can also be utilized with the SDH nucleic acid molecules disclosed in the present invention. These vectors are based on positive or negative stranded RNA viruses that naturally replicate in a wide variety of eukaryotic cells (Bredenbeek, PJ and Rice, CM, Virology 3: 297-310 ( 1992)
). Unlike retroviruses, these viruses lack an intermediate DNA life cycle and exist entirely in RNA form. For example, alphaviruses are used as expression vectors for foreign proteins. Because they can be used in a wide variety of host cells and provide high levels of expression; examples of this type of virus include Sindbis virus and Semliki Forest virus (Schlesinger, S., et al. TIBTECH 11: 18-22 (
1993); Proc. Natl. Acad. Sci.
(USA) 93: 11371-11377 (1996)). Inv
Researchers can conveniently maintain the recombinant molecule in a DNA form (pSinrep5 plasmid) in the laboratory, as exemplified by the Strogen Sinbis expression system, but propagation in RNA form is also feasible. In the host cell used for expression, the vector containing the gene of interest is completely present in RNA form, and can be grown continuously in that state, if desired.

In another embodiment, the invention further provides a variant nucleic acid molecule that encodes a portion, analog or derivative of an isolated nucleic acid molecule described herein.
Variants include those produced by nucleotide substitutions, deletions or additions, which may include one or more nucleotides. Variants may have altered coding regions, non-coding regions, or both. Changes in the coding region can result in conservative or non-conservative amino acid substitutions, deletions or additions.

Variants of the isolated nucleic acid molecules of the present invention can occur naturally (eg, naturally occurring allelic variants). By "allelic variant" is intended one of several alternative forms of a gene that occupies a given locus on the chromosome of an organism. Ge
nes II, Lewin, B .; Hen, John Wiley & Sons, N
ew York (1985). Non-naturally occurring variants can be produced using mutagenesis techniques known in the art.

[0053] The isolated nucleic acid molecules of the present invention can also be polymeric that are 95%, 96%, 97%, 98% and 99% identical to the isolated nucleic acid molecules described herein. Includes nucleotide sequence. Computer programs (for example, BestFit program (Wisconsin Sequence Analysis Packa)
ge, Version 10 for Unix®, Genetic
s Computer Group, University Research
Park, 575 Science Drive, Madison, WI 5
3711)), any particular nucleic acid molecule is at least 95%, 96% relative to the nucleotide sequence disclosed herein or the nucleotide sequence of the deposited clone described herein. , 97%, 98% or 99% identical. BestFit is available from Smith and Waterman
, Advances in Applied Mathematicals 2: 4
82-489 (1981) using the local homology algorithm to find the best segment of homology between the two sequences.

As an example, a computer alignment program (eg, BestFi
When t) is used to determine 95% identity to a reference nucleotide sequence, the percent identity is calculated over the entire length of the reference nucleotide sequence, and gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are determined. Permissible. Thus, 95% identity per 100 base pairs analyzed indicates that as much as 5 out of 100 nucleotides in the subject sequence can vary from the reference nucleotide sequence.

The present invention also includes the nucleotide sequences described herein and fragments of the isolated nucleic acid molecules. In a preferred embodiment, the invention provides fragments that are at least 20 bases in length. The length of such a fragment can be easily defined algebraically. For example, SEQ ID NO: 1 provides an isolated nucleotide molecule that is 2,265 bases in length. A fragment (F1) of SEQ ID NO: 1 that is at least 20 bases in length can be defined as F1 = 20 + X, where X is 0
Or, it is defined as any one of whole integers of 1-2,245. Similarly, fragments for other isolated nucleic acid molecules described herein may be defined as follows: for SEQ ID NO: 2, which is 1,437 bases long, A fragment (F2) of at least 20 bases in length may be defined as F2 = 20 + X, where X is defined to be either 0 or an entire integer from 1 to 1,417; 921 bases in length For SEQ ID NO: 3, a fragment (F3) of SEQ ID NO: 3 that is at least 20 bases in length can be defined as F3 = 20 + X, where X is either 0 or an entire integer from 1 to 901. Stipulated; 4,
For SEQ ID NO: 7, which is 830 bases long, a fragment of SEQ ID NO: 7 that is at least 20 bases long (F7) can be defined as F7 = 20 + X, where X is 0
Or any of the whole integers from 1 to 4,810; and 2,
For SEQ ID NO: 8, which is 700 bases long, a fragment (F8) of SEQ ID NO: 8 that is at least 20 bases long can be defined as F8 = 20 + X, where X is 0
Or, it is defined as any one of whole integers of 1 to 2,680. As will be appreciated by those skilled in the art, an isolated nucleic acid sequence fragment of the present invention can be a single-stranded or double-stranded molecule.

The present invention discloses an isolated nucleic acid sequence encoding the three subunit proteins of the SDH enzyme of the present invention. Computer analysis provides information on the open reading frame, putative signal sequence and mature protein form of each subunit. The first (75 kDa) subunit and the second (50 kD)
a) The gene encoding the subunit is a Korean Collection.
for Type Cultures (KCTC), Korea Research
rch Institute of Bioscience and Biot
technology (KRIBB), 52, Own-Dong, Yusong-
Ku, Taejon 305-33, λ GEM 5- deposited at the Republic of Korea under the accession no. It is completely contained in the 5.7 kb PstI fragment of one clone. The gene for the third (29 kDa) subunit is called ClaI- # 69 and is a Korean Collection for Type Culture.
res (KCTC), Korea Research Institute o
f Bioscience and Biotechnology (KRIBB
), 52, Own-Dong, Yusong-Ku, Taejon 305-3.
3. In the Republic of Korea, registration number KCTC 0594BP
Was deposited in bacteria on March 25, 1999 and has accession number KC as DNA.
Included in the 4.5 kb long sequence deposited on April 2, 1999 at TC 0598BP.

Thus, the present invention provides an isolated nucleic acid molecule (KCTC 0597BP) carried in the novel strain KCTC 0593BP, and the present invention also provides a single strain carried in the novel strain KCTC 0594BP. The isolated nucleic acid molecule (KCTC 05
98 BP).

The 5.7 kb Pst I fragment, the sequence obtained from λGEM 5-1 is shown in FIG. 8 and is assigned SEQ ID NO: 7. The complete coding sequence for the first subunit gene is located at positions 665-2,929 (SEQ ID NO: 1), the signal sequence is located at positions 665-766, and the first subunit of SDH The coding sequence for the mature protein of position 767 to position 2,929 (SEQ ID NO: 2)
It is located in 2). The complete coding sequence for the second subunit gene is 2
, Positions 964 to 4,400 (SEQ ID NO: 2), and the signal sequence is 2,964.
And the coding sequence for the mature protein of the second subunit of SDH is located at positions 3,072-4,400 (SEQ ID NO: 23).

Thus, another embodiment of the present invention is an isolated sequence derived from the 5.7 kb Pst I fragment of λ GEM 5-1 shown in FIG. 8 and identified as SEQ ID NO: 7. Provide a nucleic acid molecule. Such isolated nucleic acid molecules include: (1) nucleotides 1-664 of SEQ ID NO: 7, identified as SEQ ID NO: 28; (2) SEQ ID NO: identified as SEQ ID NO: 29 SEQ ID NO: 7, identified as nucleotides 50-664 of (7) SEQ ID NO: 30
(4) nucleotides 150-664 of SEQ ID NO: 7, identified as SEQ ID NO: 31; (5) nucleotides 200-664 of SEQ ID NO: 7, identified as SEQ ID NO: 32; SEQ ID NO: 7 identified as nucleotides 250-664; SEQ ID NO: 34; (7) SEQ ID NO: 7 identified as nucleotides 300-664; (8) SEQ ID NO: 35 identified as SEQ ID NO: 35. (9) nucleotides 400-664 of SEQ ID NO: 7, identified as SEQ ID NO: 36; (10) SEQ ID NO: 3
(11) nucleotides 500-664 of SEQ ID NO: 7, identified as SEQ ID NO: 38;
) Nucleotides 550-664 of SEQ ID NO: 7, identified as SEQ ID NO: 39;
(13) nucleotides 600-6 of SEQ ID NO: 7, identified as SEQ ID NO: 40
64; (14) nucleotides 665 of SEQ ID NO: 7, identified as SEQ ID NO: 1
Nucleotide sequence encoding the SDH full length subunit 1 protein of the present invention; (15) nucleotides 767-2,929 of SEQ ID NO: 7, identified as SEQ ID NO: 22. Nucleotide sequence encoding the mature form of subunit 1 protein; (16) encoding the full-length SDH subunit 2 protein of the invention at nucleotides 2,964-4,400 of SEQ ID NO: 7, identified as SEQ ID NO: 2 (17) SEQ ID NO: 2
Nucleotide sequence encoding the subunit 2 protein of the mature form of SDH of the present invention at nucleotides 3,072-4,400 of SEQ ID NO: 7, identified as SEQ ID NO: 3; (18) sequence identified as SEQ ID NO: 41 Nucleotide 2 of number 7
930 to 2,963; (19) identified as SEQ ID NO: 42, nucleotides 4,401 to 4,451 of SEQ ID NO: 7; (20) identified as SEQ ID NO: 43;
SEQ ID NO: 7, nucleotides 4,401 to 4,501; (21) SEQ ID NO: 44, identified as nucleotides 4,401 to 4,551; (22) SEQ ID NO: 45, identified as SEQ ID NO: 7, nucleotides 4,401 to 4,60
1; (23) nucleotides 4, 4 of SEQ ID NO: 7, identified as SEQ ID NO: 46
(24) nucleotides 4,401 to 4,701 of SEQ ID NO: 7, identified as SEQ ID NO: 47; (25) nucleotides 4,401 to SEQ ID NO: 7, identified as SEQ ID NO: 48 (26) nucleotides 4,401-4,801 of SEQ ID NO: 7, identified as SEQ ID NO: 49; and (27)
) Nucleotides 4,401-4, of SEQ ID NO: 7, identified as SEQ ID NO: 50.
830.

The sequence obtained from the Cla I- # 69 clone is shown in FIG. 10 and is assigned SEQ ID NO: 8. The complete coding sequence for the third subunit gene is 1
, Positions 384 to 2,304 (SEQ ID NO: 3), and the signal sequence is 1,384
Located at positions １，1,461 and the coding sequence for the mature protein of the third subunit of SDH is at positions 1,462-2,304 (SEQ ID NO: 24).

Accordingly, another embodiment of the present invention provides an isolated nucleic acid molecule derived from the sequence obtained from the Cla I- # 69 clone, exemplified in FIG. 10 and assigned SEQ ID NO: 8. provide. Such isolated nucleic acid molecules include: (1) nucleotide 1 of SEQ ID NO: 8, identified as SEQ ID NO: 51
(2) nucleotides 50-1,383 of SEQ ID NO: 8, identified as SEQ ID NO: 52; (3) nucleotides 100-1,383 of SEQ ID NO: 8, identified as SEQ ID NO: 53; 4) nucleotides 150-1 383 of SEQ ID NO: 8, identified as SEQ ID NO: 54; (5) identified as SEQ ID NO: 55,
(6) nucleotides 250 to 1,383 of SEQ ID NO: 8, identified as SEQ ID NO: 56; (7) nucleotides 300 of SEQ ID NO: 8, identified as SEQ ID NO: 57. (8) nucleotides 350-1,383 of SEQ ID NO: 8, identified as SEQ ID NO: 58;
) Nucleotides 400-1, 38 of SEQ ID NO: 8, identified as SEQ ID NO: 59
3; (10) nucleotide 450 of SEQ ID NO: 8, identified as SEQ ID NO: 59
(11) nucleotides 500 to 1,383 of SEQ ID NO: 8, identified as SEQ ID NO: 61; (12) SEQ ID NO: 8 identified as SEQ ID NO: 62
(13) identified as SEQ ID NO: 63;
(14) nucleotides 600 to 1,383 of SEQ ID NO: 8, identified as SEQ ID NO: 64; (15) SEQ ID NO: 6
Nucleotides 650-1,383 of SEQ ID NO: 8, identified as 5; (16)
Nucleotides 700 to 1,383 of SEQ ID NO: 8, identified as SEQ ID NO: 66
(17) nucleotides 750-750 of SEQ ID NO: 8, identified as SEQ ID NO: 67;
(18) nucleotides 800-1383 of SEQ ID NO: 8, identified as SEQ ID NO: 68; (19) nucleotides 850-1,383 of SEQ ID NO: 8, identified as SEQ ID NO: 69; ) Nucleotides 900-1, 383 of SEQ ID NO: 8, identified as SEQ ID NO: 70; (21) nucleotides 950-1 383 of SEQ ID NO: 8, identified as SEQ ID NO: 71; (22) SEQ ID NO: 72.
SEQ ID NO: 8 nucleotides 1,000-1,383; (23
) Nucleotides 1,050-1 of SEQ ID NO: 8, identified as SEQ ID NO: 73;
383; (24) nucleotide 1 of SEQ ID NO: 8, identified as SEQ ID NO: 74
(25) nucleotides 1,150 to 1,383 of SEQ ID NO: 8, identified as SEQ ID NO: 75; (26) nucleotides 1,200 of SEQ ID NO: 8, identified as SEQ ID NO: 76. (27) nucleotides 1,250-1,383 of SEQ ID NO: 8, identified as SEQ ID NO: 77; (28)
Nucleotides 1,300 to 1,3 of SEQ ID NO: 8, identified as SEQ ID NO: 78
83; (29) nucleotide 1, SEQ ID NO: 8, identified as SEQ ID NO: 79
(30) the nucleotide sequence of nucleotides 1,384 to 1,461 of SEQ ID NO: 8, encoding the full length SDH subunit 3 protein of the present invention, identified as SEQ ID NO: 3; Identified as number 24,
8. A mature form of the SD of the invention at nucleotides 1,462-2,304 of SEQ ID NO: 8
Nucleotide sequence encoding the H subunit 3 protein; (32) nucleotides 2,305-2,355 of SEQ ID NO: 8, identified as SEQ ID NO: 80;
33) Nucleotides 2,305-SEQ ID NO: 8, identified as SEQ ID NO: 81
2,405; (34) nucleotides 2,305-5,455 of SEQ ID NO: 8, identified as SEQ ID NO: 82; (35) nucleotides 2,305-5,2 of SEQ ID NO: 8, identified as SEQ ID NO: 83. 505; (32) nucleotides 2,305-2,555 of SEQ ID NO: 8, identified as SEQ ID NO: 84; (32) SEQ ID NO: 8
Nucleotides 2,305-2,605 of SEQ ID NO: 8, identified as SEQ ID NO: 5; (3
2) Nucleotides 2,305-2 of SEQ ID NO: 8, identified as SEQ ID NO: 86
655; and (33) nucleotides 2,305-2,700 of SEQ ID NO: 8, identified as SEQ ID NO: 87.

The present invention also includes a recombinant construct comprising one or more sequences as broadly described above. The construct includes a vector (eg, a plasmid or viral vector) into which the sequences of the invention have been inserted in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises a regulatory sequence operably linked to the sequence (eg, including a promoter). Many suitable vectors and promoters are known in the art and are commercially available. The following vectors are provided as examples: bacterial-pET (Novagen), pQE70, p
QE60, pQE-9 (Qiagen), pBs, phasescript, p
siX174, pBlueScript SK, pBsKS, pNH8a, pN
H16a, pNH18a, pNH46a (Stratagene); pTrc9
9A, pKK223-3, pKK233-3, pDR540, pRIT5 (Ph5
and eukaryotic-pWLneo, pSV2cat, pOG
44, pXT1, pSG (Stratagene) pSVK3, pBPV, pM
SG, pSVL (Pharmacia). Thus, these and any other plasmids or vectors can be used as long as they are replicable and viable in their host.

The promoter region can be selected from any desired gene using a CAT (chloramphenicol transferase) vector, or other vector having a selectable marker. Two suitable vectors are pKK232-8 and pCM7
It is. Specific designated bacterial promoters include lacI, lacZ, T
3, T7, gpt, λP _R , P _L and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV4
0, retrovirus-derived LTR and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of skill in the art.

In another embodiment, the present invention provides a process for producing a vector described herein, comprising: (a) isolating the present invention. Inserting the nucleic acid molecule into a vector; and (b) selecting and growing the vector in a host cell.

Representative examples of suitable hosts include, but are not limited to: bacterial cells (eg, Gluconobacter, Brevibacteriu)
m, Corynebacterim, E.M. coli, Streptomyces
, Salmonella typhimurium, Acetobacter,
Pseudomonas, Pseudogluconobacter, Baci
llus and Agrobacterium cells); fungal and yeast organisms (S
accaromyces, Kluyveromyces, Aspergill
and Rhizopus); insect cells (eg, Drosophil)
a cells of S2 and Spodoptera Sf9); animal cells (eg, C
HO, COS and Bowes melanoma cells); and plant cells. Suitable culture media and conditions for the above host cells are known in the art.

(4. Polypeptide of the Present Invention) The present invention provides an isolated polypeptide molecule for the SDH enzyme of the present invention. Methods and techniques designed for the manipulation of isolated polypeptide molecules are well known in the art. For example, methods for the isolation and purification of polypeptide molecules are described in Current Protocols in Protein Sci.
ence, John E. Ed. Coligan et al., John Wiley & S
ons, Inc. , 1997, the disclosure of which is incorporated herein by reference.

More particularly, the present invention provides a number of isolated polypeptide molecules that encode the individual 75 kDa, 50 kDa and 29 kDa subunit proteins of the SDH enzymes of the present invention. For purposes of clarity, certain isolated polypeptide molecules of the present invention are described. Thereafter, the specific properties and characteristics of these isolated polypeptide molecules are described in more detail.

In one embodiment, the present invention provides an isolated polypeptide comprising a polypeptide sequence selected from the group consisting of: (a) encoded in the polynucleotide sequence of SEQ ID NO: 1 (B) the polypeptide sequence of SEQ ID NO: 4; and (c) at least about 10 polypeptide sequences derived from the polypeptide sequence of SEQ ID NO: 4.
An amino acid long polypeptide.

[0069] In another embodiment, the present invention provides an isolated polypeptide comprising a polypeptide sequence selected from the group consisting of: (a) a polynucleotide encoded by the polynucleotide sequence of SEQ ID NO: 2. (B) the polypeptide sequence of SEQ ID NO: 5; and (c) at least about 10 polypeptide sequences derived from the polypeptide sequence of SEQ ID NO: 5.
An amino acid long polypeptide.

In yet another embodiment, the invention provides an isolated polypeptide comprising a polypeptide sequence selected from the group consisting of: (a) in the polynucleotide sequence of SEQ ID NO: 3 An encoded polypeptide sequence; (b) a polypeptide sequence of SEQ ID NO: 6; and (c) a polypeptide of at least about 10 amino acids in length derived from the polypeptide sequence of SEQ ID NO: 6.

Another embodiment of the present invention includes an isolated polypeptide sequence comprising a polypeptide encoded by the isolated nucleic acid sequence SEQ ID NO: 7; an isolated nucleic acid sequence SEQ ID NO: An isolated polypeptide sequence comprising the polypeptide encoded by the DNA clone contained in KCTC Accession No. 0593BP; and KC
Isolated polypeptide sequences including the polypeptide encoded by the DNA clone contained in TC Accession No. 0594BP.

The term “isolated polypeptide” is used herein to mean a polypeptide that has been removed from its native environment. Thus, polypeptides produced and / or contained in a recombinant host cell are considered isolated for the purposes of the present invention. An “isolated polypeptide” also intends a polypeptide that is partially or substantially purified from a recombinant host cell.

The polypeptides of the present invention include natural purified products, products of chemical synthetic procedures, and prokaryotic or eukaryotic hosts, such as bacterial cells, yeast cells, higher plant cells, insect cells and mammalian cells. (Including animal cells) produced by recombinant techniques. Depending on the host used in the recombinant production procedure,
The polypeptides of the present invention may be glycosylated or non-glycosylated. Further, the polypeptides of the present invention may also contain an initial modified methionine residue, in some cases as a result of a host-mediated process.

[0074] Isolated polypeptides of the present invention also include variants of the above-described polypeptides. The term "variant" is meant to include the naturally occurring allelic variant polypeptide sequences that carry conservative or non-conservative substitutions, deletions or insertions of amino acids. The term "variant" is also meant to include an isolated polypeptide sequence produced by a human hand through known mutagenesis techniques or through chemical synthesis methodology. Such artificial variants may include polypeptide sequences that carry conservative or non-conservative substitutions, deletions or insertions of amino acids.

[0075] Whether a particular amino acid is conservative or non-conservative is well known to those of skill in the art. Conservative amino acid substitutions have no significant effect on protein folding or activity. For illustrative purposes, Table 1 shows a list of conservative amino acid substitutions.

[0076]

[Table 1] Amino acids essential for function in the protein of the present invention can be obtained by methods known in the art (for example, site-directed mutagenesis or alanine scanning mutagenesis (Cu
nningham and Wells, Science 244: 1081-10.
85 (1989)).

The isolated polypeptide molecules of the present invention also include polypeptides that are 95%, 96%, 97%, 98% and 99% identical to the isolated polypeptide molecules described herein. Peptide sequences. Computer programs (eg Bes
tFit program (Wisconsin Sequence Analysis)
s Package, Version 10 for Unix (registered trademark),
Genetics Computer Group, University R
search Park, 575 Science Drive, Madis
On, WI 53711)), any particular polypeptide molecule can be isolated from a polypeptide sequence disclosed herein or an isolated DNA molecule of a deposited clone described herein. May be determined to be at least 95%, 96%, 97%, 98% or 99% identical to the polypeptide sequence encoded by BestFit is available from Smith and Waterman, Adv
ances in Applied Matrices 2: 482-4
89 (1981) using the local homology algorithm to find the best segment of homology between the two sequences.

As an example, a computer alignment program (eg, BestFi
When t) is used to determine 95% identity to a reference polypeptide sequence, the percentage identity is calculated over the entire length of the reference polypeptide sequence and in homology up to 5% of the total number of amino acids in the reference sequence. Gaps are allowed. Thus, for every 100 amino acids analyzed, 95% identity indicates that as much as 5 out of 100 amino acids in the subject sequence can vary from the reference polypeptide sequence.

The present invention also encompasses polypeptide sequences described herein and fragments of isolated polypeptide molecules. In a preferred embodiment, the present invention provides
Provide fragments that are at least 10 amino acids in length. The length of such a fragment can be easily defined algebraically. For example, SEQ ID NO: 4 provides an isolated polypeptide molecule that is 754 amino acids long. A fragment of SEQ ID NO: 4 that is at least 10 amino acids in length (F4) may be defined as F4 = 10 + X,
Here, X is defined to be either 0 or an entire integer from 1 to 744.
Similarly, fragments for other isolated polypeptide molecules described herein can be defined as follows: SEQ ID NO: 5 which is 478 amino acids long
For a fragment of SEQ ID NO: 5 which is at least 10 amino acids in length (F5)
Can be defined as F5 = 10 + X, where X is defined as either 0 or an entire integer from 1 to 468; and for SEQ ID NO: 6 which is 306 amino acids long, A fragment (F6) that is at least 10 amino acids in length can be defined as F6 = 10 + X, where X is defined to be either 0 or an entire integer from 1 to 296.

A particularly preferred embodiment of the present invention provides an isolated polypeptide as follows: (1) encoded by the isolated nucleic acid molecule of SEQ ID NO: 1 and identified by SEQ ID NO: 4 (2) the full-length polypeptide of SDH subunit 2 of the present invention encoded by the isolated nucleic acid molecule of SEQ ID NO: 2 and identified by SEQ ID NO: 5 peptide;
(3) encoded by the isolated nucleic acid molecule of SEQ ID NO: 3 and SEQ ID NO: 6
A full-length polypeptide of SDH subunit 3 of the present invention, identified by (4)
) Encoded by the isolated nucleic acid molecule of SEQ ID NO: 22, and
Mature form of the SDH subunit 1 polypeptide of the invention, identified by:
(5) a mature form of the SDH subunit 2 polypeptide of the invention, encoded by the isolated nucleic acid molecule of SEQ ID NO: 24 and identified as SEQ ID NO: 26; and the isolated nucleic acid molecule of SEQ ID NO: 23 A mature form of the SDH subunit 3 polypeptide of the invention, encoded by and identified as SEQ ID NO: 27.

The present invention also provides a process for producing a polypeptide, comprising the steps of: (a) comprising an isolated nucleic acid molecule of SEQ ID NO: 1 or a variant thereof. Growing a host cell; (b) expressing a polypeptide encoded by the isolated nucleic acid molecule; and (c) isolating the polypeptide.

In another embodiment, the invention provides a process for producing a polypeptide, comprising the steps of: (a) isolated nucleic acid molecule of SEQ ID NO: 2 or Growing a host cell containing the variant; (b) expressing a polypeptide encoded by the isolated nucleic acid molecule; and (c).
Isolating the polypeptide.

Another process provided by the present invention is a process for producing a polypeptide, comprising the following steps: (a) isolated nucleic acid molecule of SEQ ID NO: 3 or Growing a host cell containing the variant; (b) expressing the polypeptide encoded by the isolated nucleic acid molecule; and (c) isolating the polypeptide.

[0084] Representative examples of suitable hosts include, but are not limited to: bacterial cells (eg, Gluconobacter, Brevibacteriu)
m, Corynebacterim, E.M. coli, Streptomyces
, Salmonella typhimurium, Acetobacter,
Pseudomonas, Pseudogluconobacter, Baci
llus and Agrobacterium cells); fungal and yeast organisms (S
accaromyces, Kluyveromyces, Aspergill
and Rhizopus); insect cells (eg, Drosophil)
a cells of S2 and Spodoptera Sf9); animal cells (eg, C
HO, COS and Bowes melanoma cells); and plant cells. Suitable culture media and conditions for the above host cells are known in the art.

The polypeptide may be expressed in a modified form (eg, as a fusion protein) and may contain not only secretion signals but also additional heterologous functional regions. For example, regions of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to provide stability in host cells during purification or during subsequent handling and storage. Can improve gender and persistence. Also, a peptide moiety can be added to the polypeptide to facilitate purification. Such regions can be removed prior to final preparation of the polypeptide. Among other things, the addition of peptide moieties to polypeptides that result in secretion or excretion, improve stability, and facilitate purification is well known in the art and is a conventional technique.

The polypeptides of the invention can be recovered and purified from recombinant cell culture by well-known methods, including: ammonium sulfate precipitation or ethanol precipitation, acid extraction, anion exchange or cation exchange chromatography,
Phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is used for purification.

5. Production of L-sorbose and 2-keto-L-gulonic acid The present invention provides a process for the production of L-sorbose and 2-keto-L-gulonic acid, which comprises: Useful for vitamin C production.

In one embodiment, the present invention provides a process for the production of L-sorbose from D-sorbitol, comprising the following steps: (a)
Selected from the group consisting of: (i) a polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 1; (ii) a polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 2; and (iii) a polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 3. Transforming a host cell with at least one isolated nucleotide sequence to be performed; and (b) selecting and growing the transformed host cell.

In another embodiment, the present invention provides a process for the production of 2-keto-L-gulonic acid, comprising the following steps: (a) (i) SEQ ID NO: 1
At least one selected from the group consisting of: (ii) a polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 2; and (iii) a polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 3.
Transforming a host cell with the two isolated nucleotide sequences; and (b)
A) selecting and growing the transformed host cells;

Suitable bacteria for use as host cells in the processes applied herein for the production of L-sorbose and 2-keto-L-gulonic acid are known to those skilled in the art. Such bacteria include Escherichia coli, B
rebacterium, Corynebacterium, Glucon
obactor, Acetobacter, Pseudomonas and Ps
eudogluconobacter, but not limited thereto.

Other host cells for expression of the SDH enzymes of the present invention include the following: strains identified in US Pat. No. 5,834,231; Glucanoba
cter oxydans DSM 4025 (U.S. Pat. No. 4,960,695)
No.); Gluconobacter oxydans TIOO (Appl. E
nviron. Microbiol. 63: 454-460 (1997));
pseudogluconobacter saccharoketogenes
IFO 14464 (EP 221 707); Pseudomona
s sorbosooxidans (U.S. Pat. No. 4,933,289); and Acetobacter liquefaciens IFO 12258 (A
ppl Environ. Microbiol. 61: 413-420 (199
5)).

In other embodiments of the present invention, various fermentation techniques known in the art may be used in the processes of the present invention for the production of L-sorbose and 2-keto-L-gulonic acid. Generally, L-sorbose and 2-keto-L-gulonic acid can be produced by a fermentation process (eg, of a batch or fed-batch type). In a batch fermentation, all nutrients are added at the start of the fermentation. In a fed-batch or long-term fed-batch fermentation, one or more nutrients may be obtained immediately after the start of fermentation or after the culture has reached a certain age, or when the nutrients supplied have been exhausted from the culture. Is continuously supplied to the culture. A long-term batch variant of a fed-batch fermentation is a repeat fed-batch or fill-and-draught fermentation.
aw fermentation, wherein a portion of the contents of the fermenter is withdrawn at some point (eg, when the fermenter is filled), during which time the nutrient supply is continued. In this way, the fermentation can be extended over time.

[0093] Another type of fermentation, continuous fermentation or chemostat cultivation, involves removing the culture medium continuously or semi-continuously, such that the volume of broth in the fermenter remains approximately constant. Use a continuous feed of Continuous fermentation can in principle be maintained for an infinite amount of time.

In batch fermentation, the organism is maintained until one of the essential nutrients in the medium is exhausted or the fermentation conditions are adversely affected (eg, the pH is reduced to a value that is inhibitory to microbial growth). (Until it drops). In feed-batch fermentations, measurements are usually taken to maintain favorable growth conditions, for example by using a pH control, and depletion of one or more essential nutrients supplies these nutrients to the culture. Is prevented by The microorganism continues to grow at a growth rate dictated by the nutrient supply rate. In general, a single nutrient (very often a carbon source) is a limiting factor for growth. The same principle applies to continuous fermentation, where one nutrient in the medium feed is usually the limiting factor,
All other nutrients are in excess. Restrictive nutrients are present at very low concentrations in the culture and are often too low to measure. Different types of nutrient restrictions can be used. Carbon source limitations are most often used. Other examples are nitrogen source limitations,
Limitation by oxygen, limitation by specific nutrients (eg, vitamins or amino acids (if the microorganism is auxotrophic for such compounds), limitation by sulfur, and limitation by phosphorus.

Illustrative examples of suitable supplemental carbon sources include, but are not limited to, other carbohydrates such as glucose, fructose, mannitol, starch or starch hydrolysates, cellulose hydrolysates and molasses Organic acids such as acetic, propionic, lactic, formic, malic, citric and fumaric acids; and alcohols such as glycerol.

Illustrative examples of suitable nitrogen sources include, but are not limited to, ammonia (including ammonia gas and aqueous ammonia); ammonium salts of inorganic or organic acids (eg, ammonium chloride, ammonium nitrate) Urea; nitrates or nitrites, and other nitrogen-containing materials (amino acids, either as pure or crude preparations, meat extracts, peptones, fish meal, fish water) Decomposition products, corn steep liquor, casein hydrolyzate, soybean cake hydrolysate, yeast extract, dried yeast, ethanol-yeast distillate, soybean powder, cottonseed powder, etc. Including).

Illustrative examples of suitable inorganic salts include, but are not limited to: potassium, calcium, sodium, magnesium, manganese, iron, cobalt, zinc, copper and other trace elements, and phosphoric acid Salt.

Illustrative examples of suitable micronutrients, growth factors and the like include, but are not limited to: as pure or partially purified chemical compounds or as present in natural materials Any of coenzyme A, pantothenic acid, biotin, thiamine, riboflavin, flavin mononucleotide, flavin adenine dinucleotide, other vitamins, amino acids (eg, cysteine), sodium thiosulfate, p-aminobenzoic acid, niacinamide Such. Cultivation of the microbial strains of the invention can be accomplished using any of the submerged fermentation techniques known to those of skill in the art, such as aeration, traditional agitated design or shaking culture.

The culturing conditions used (including temperature, pH, aeration rate, agitation speed, culturing duration, etc.) depend on one skilled in the art to maximize the production of L-sorbose and 2-keto-L-gulonic acid. Can be determined empirically. The choice of particular culture conditions will depend on factors such as the particular microbial strain of the invention used, the medium composition and type, the culture technique, and similar considerations.

An illustrative example of a suitable method for recovering 2-KLG is US Pat.
Nos. 74,924; 5,312,741; 4,960,695; 4,935,359; 4,877,735; 4,933,289; No. 4,892,823; No. 3,043,749; No. 3,912,5
No. 92; 3,907,639 and 3,234,105.

According to one such method, the microorganism is firstly removed from the culture broth by known methods (eg, centrifugation or filtration), and the resulting solution is treated under reduced pressure. It is concentrated. The crystalline 2-KGL is then recovered by filtration and, if desired, purified by recrystallization. Similarly, 2-KGL can be recovered using known methods such as using ion exchange resins, solvent extraction, precipitation, salting out, and the like.

When 2-KGL is recovered as the free acid, the free acid can be converted to a salt with sodium, potassium, calcium, ammonium or a similar cation, if desired, using conventional methods. Alternatively, if 2-KGL is recovered as a salt, the salt can be converted to its free form or to a different salt using conventional methods.

[0103] All patents and publications mentioned herein are expressly incorporated by reference. Although the present invention has been described generally, the present invention can be more readily understood by reference to the following examples. It should be noted that the following examples are provided for illustrative purposes and are not intended to limit the invention unless otherwise indicated.

EXAMPLES Example 1 Isolation and Characterization of SDH from G. suboxydans KCTC 2111 An improved method of purifying pyrroquinoline quinone (PQQ) -dependent SDH of the present invention from suboxydans KCTC 2111 is shown. Initial purification scheme (Choi, ES, et al., FEMS Microbiol. Lett.
. , 125: 45 (1995)) relates to greater subunit resolution and improved stability of enzyme activity.

(Step 1: Culture of G. suboxydans KCTC 2111) suboxydans KCTC 2111 was added to 5 ml of SYP medium (5
% D-sorbitol, 1% BactoPeptone and 0.5% yeast extract) and incubated at 30 ° C. for 20 hours. Transfer 1 milliliter (ml) of this culture to 50 ml of the same medium in a 500 ml flask,
The cells were cultured at 30 ° C. for 20 hours using a rotary shaker (180 rpm). The culture thus prepared was combined with a 5 L jar fermenter (jar) containing 3 L of the same medium.
fermentor) and a 3 L culture was
Propagated until early stationary phase.

(Step 2: Preparation of Membrane Fraction) Cells were collected by centrifugation at 12,000 g for 10 minutes, washed once with 10 mM sodium acetate buffer (pH 5.0), and bead beater (bead)
beater (Edmund Buhler, Vi 4) at 4 ° C.
Broken for 2 seconds using glass beads (0.1 mm diameter). The homogenate thus prepared was centrifuged at 12,000 g for 5 minutes to remove cell debris and glass beads. The obtained supernatant was centrifuged at 100,000 g for 60 minutes, and a crude membrane fraction was obtained as a precipitate.

(Step 3: Solubilization of SDH from membrane fraction) The crude membrane fraction was resuspended at 40 mg protein / ml and stirred at 4 ° C for 2 hours to give 1.5% n- Solubilized with octyl glucoside. The obtained suspension was centrifuged at 12,000 g for 10 minutes to obtain a supernatant, which was referred to as a solubilized SDH fraction. All solutions used in the purification procedure were 0.7
It contained 5% n-octylglucoside.

Step 4: Enzyme activity assay Enzyme activity was determined using 2,6-dichlorophenol iodophenol (DCIP) as an artificial electron acceptor and phenazine methosulfate (phenazi)
nemethosulfate (PMS) as the electron mediator was assayed spectrophotometrically. This reaction mixture is mixed with 50 ml in a total volume of 1.0 ml.
mM sodium acetate buffer (pH 5.0), 10 mM MgCl ₂ , 5 mM C
aCl ₂ , 5 mM KCN, 0.1 mM PMS, 0.12 mM DCIP, 2
It contained 50 mM D-sorbitol, 0.75% n-octylglucoside and enzyme solution. A molar extinction coefficient of ε = 5,600 cm ⁻¹ M ⁻¹ for DCIP at pH 5.0 was used. One unit of enzyme activity is converted to 1 μmol of DC per minute.
It was defined as the amount of enzyme that catalyzes the reduction of IP.

Enzyme activity was determined for the reconstituted subunit described in Step 6 of Example 1 by the Ferric-Dupanol method (Wood, WA, et al., Meth. En.
zymol. 5: 287 (1962)). The subunit proteins were preincubated for 5 minutes at 25 ° C., either alone or in different combinations. The reaction was started by the addition of 10 mM (final concentration) potassium ferricyanide and 250 mM (final concentration) D-sorbitol. After an appropriate time, the reaction is carried out with a ferrous sulfate-Dupanol solution (F
Stop by adding e ₂ (SO ₄ ) ₃ .nH ₂ O 5 g / L, Dupanol (sodium lauryl sulfate) 3 g / L and 85% phosphoric acid 95 ml / L)
The Prussian absorbance was then determined at 660 nm on a spectrophotometer.

(Step 5: Ion Exchange Chromatography) The solubilized fraction was equilibrated with 10 mM sodium acetate (pH 5.0) to a CM-TSK 650 (S) (Merck) column (2.5 × 20 cm). To The CM-TSK column was eluted with a linear gradient (from 10 mM to 500 mM) of sodium acetate. Active fractions were pooled and concentrated by ultrafiltration using a membrane filter (Amicon, YM 10) and DEA
It was applied to an E-TSK 650 (S) (Merck) column (2.5 × 20 cm). The DEAE-TSK column was loaded with either pH 5.0 or pH 6.0.
Isocratically with 0 mM sodium acetate buffer
ly) eluted.

As shown in FIG. 1, when eluted at pH 6.0 (FIG. 1-B), the resolution of the activity peak is much higher than at pH 5.0 (FIG. 1-A), and the enzyme activity Was completely separated from the peaks I and II. Peaks I and II co-eluting at pH 5.0 when the enzyme activity of the fractions was measured immediately after elution and 1 hour after elution.
It was found that the enzymatic activity of the fractions in did not differ significantly over time.
However, for fractions in peaks I and II that elute separately at pH 6.0,
The enzyme activity in peak I was reduced to less than one tenth of its original value within one hour of elution, while the enzyme activity in peak II remained unchanged.

On a DEAE-TSK column, the fraction containing peaks I and II eluted at pH 6.0 was subjected to SDS-PAGE (Laemmli, UK, Nature, 22
7, 680 (1970)) (FIG. 2). FIG. 2-A shows the fraction of peak I. Column M shows standard molecular weight markers. As shown in columns 1-4, a first subunit band of 75 kDa and a second subunit band of 50 kDa
Can be observed, but the 29 kDa third subunit band was not observed. In this case, after 1 hour of elution, the enzyme activity decreased by more than 10-fold. FIG. 2-B shows the fraction of peak II. Column M shows standard molecular weight markers. As shown in columns 5 to 8, the fraction of peak II was 7
It was observed to contain a third subunit band of 29 kDa in addition to the 5 kDa and 50 kDa subunits. This observation is 29k
It has been shown that the third subunit of Da may play an important role in the stability of SDH.

Step 6: SDH Reconstitution with Different Subunits Increasing the pH of the elution buffer from 5.0 to 6.0 during DEAE column chromatography increased the resolution of the chromatographic peaks Therefore, the pH of the elution buffer was further raised to pH 6.5. As described in step 5 of Example 1, partially purified SDH provided by CM-TSK 650 (S) column chromatography was prepared by pre-equilibrating DEAE- TSK 650 (S) column (2.5 x 16.5 cm)
And the column was eluted isocratically with 180 ml of the same buffer, followed by a gradient elution with 160 ml each of 20 mM and 500 mM sodium phosphate buffer, pH 6.5. Column fractions were analyzed by SDS-PAGE.

As shown in FIG. 3, the 75 kDa first subunit elutes first (peak I), followed by the 29 kDa third subunit eluted during the isocratic elution (peak II). And a 50 kDa second subunit eluted later during the gradient elution (peak III). As shown in FIG. 4, the SD of these three peaks
S-PAGE analysis showed that the three subunits were essentially pure and completely separated from each other.

As the three subunits can be purified under non-denaturing conditions and separated into individual species, reconstitution experiments were performed to determine the role of each subunit on the catalytic activity of the SDH enzyme. Approximately 100 pmole of each subunit obtained from the DEAE column by elution at pH 6.5, alone or in a different combination, was pre-incubated and, as described in Example 1, step 4, Ferric-
Enzyme activity was assayed by the Dupanol method (Table 2). Each subunit alone exhibited very low levels of activity. When the first subunit and the second subunit were reconstituted, the enzymatic activity increased two-fold compared to the first subunit alone. When the third subunit is added to the first subunit, and when added to a mixture of the first subunit and the second subunit, the enzyme activity is about 30-fold and about 30-fold, respectively. Up 20 times. These results indicated that the third subunit plays an important role in the catalytic activity of SDH as well as the stability of this enzyme.

[0116]

[Table 2] (Example 2: Determination of N-terminal amino acid sequence of SHD subunit) Purified SDH prepared in Example 1 was subjected to SDS-PAGE (12.5
% Gel) and the separated proteins are loaded on polyvinylidene difluoride (
PVDF) membranes (Bollag, DM and Edelstein, S.M.
J. , Protein Methods, Wiley-Liss, Inc. , 8
(1991)). After visualization with Ponceau S dye, the portion of the membrane containing each SDH subunit was cut into small pieces, and the small pieces of this membrane for each subunit were analyzed using an amino acid sequence analyzer (Ap
plied Biosystems, Model 477A) was applied directly for N-terminal amino acid sequence analysis.

Table 3 shows the data obtained for the first and third subunits.
(SEQ ID NO: 9 and SEQ ID NO: 11). The N-terminal amino acid sequence of the second subunit could not be obtained. This is most likely due to the N-terminus being blocked. Similar findings for the blocked N-terminus were found in another acetic acid bacterium, Acet.
report on the cytochrome c subunit of the alcohol dehydrogenase complex of O. pasteurianus (Takemura, H
. Et al., J Bacteriol. 175, 21, 6857 (1993)), wherein the N-terminal block was due to a modification of a glutamic acid residue at the N-terminus to a pyroglutamic acid residue. This modification resists Edman degradation during N-terminal amino acid sequencing.

Therefore, to obtain the N-terminal sequence for the second subunit of purified SDH, the isolated SDH protein was first treated with pyroglutamate aminopeptidase to potentially block it. The N-terminus was released. Briefly, about 50 pg of purified SDH from Example 1 was added to digestion buffer (100 mM
Dissolved in sodium phosphate buffer, 10 mM EDTA, 5 mM dithiothreitol and 5% (v / v) glycerol, pH 8.0) and 3.75 μg of pyroglutamic aminopeptidase (Boehringer Mannhei).
m) for 18 hours at 4 ° C., followed by a further 4 hours at 25 ° C. After incubation, the reaction mixture was subjected to SDS-PAGE, electroblotted onto a PVDF membrane, and the second subunit band on the membrane was excised and analyzed for N-terminal amino acid sequence as described above. The N-terminal amino acid sequence of 10 residues is shown in Table 3 as SEQ ID NO: 10.

[0119]

[Table 3] Example 3 Determination of the Internal Amino Acid Sequence for the Third Subunit For the third subunit, the internal amino acid sequence was also determined in addition to the N-terminal amino acid sequence for enhanced cloning of the corresponding gene. did. Example 1
Approximately 7 μg of the third subunit protein, isolated as described in step 6 of, was previously described (Matsudaira, PT, A Practica).
l Guide to Protein and Pept Purifica
Tion for Microsequencing Academic Pre
ss page 37 (1989)) and trypsin (Boehringer Ma).
nnheim). The trypsinized digest was purchased from Brownlee SP
Separation by HPLC using a HERI-5 RP 18 column (0.2 × 22 cm) and separating the column with a linear gradient of 15-70% acetonitrile over 21%
Elution was performed at 0 microliter / min for 60 minutes. Elution at 214 nm
And three well-resolved peptide peaks were collected (designated 1, 5 and 7 in FIG. 5) and analyzed for their amino acid sequence as described in (Example 2). The internal amino acid sequences for peaks 1, 5, and 7 are shown in Table 4 below (SEQ ID NO: 12, SEQ ID NO: 13, respectively)
And SEQ ID NO: 14).

[0120]

[Table 4] (Example 4: Primer design and 1.53 kb including part of SDH gene)
Isolation of DNA Fragment) Based on the N-terminal amino acid sequence of the first subunit (SEQ ID NO: 9), two degenerate primers, Primer 1 (5'-CCGGAATTC GAA (G)
GAT (C) ACI GGI ACI GC-3 ') (SEQ ID NO: 15) and primer 2 (5'-ATT (C, A) ACI AAT (C) GCI GAT (C
) CAAG) CAT (C) CC-3 ') (SEQ ID NO: 16) was synthesized.

Takeda and Shimizu (Takeda and Shimizu, J
. Ferm. Bioeng. , 72: 1 (1991)). su
genomic DNA isolated from B. boxydans KCTC 2111 was purified from Bam
Partially digested with HI. Plasmid pBluescript SK (Strata
Tagene) was restricted with NaeI and BamHI and genomic DN
A BamHI partial digest of A was ligated into the BamHI site of this plasmid.

[0122] Thirty cycles of the polymerase chain reaction (PCR) were performed using the monospecific primer P
CR (SSP-PCR) method (White, B., SSP-PCR and g
enome walking, Method in Mol. Biol. , PC
R Protocol, Humana Press, 15: 339 (1993)
) To isolate a clone (# SDH2-1) with a size of 1.53 kb.

The ligation reaction mixture prepared above was amplified with primer 1, a gene-specific primer, and the T7 primer of this vector. The generic T7 primer anneals to the ends of all ligated fragments, but the resulting product increases only linearly. However, simultaneous annealing of gene specific primer 1 and T7 primer to a specific product results in exponential amplification of the specific primary product. Gene-specific primer 2 which is a nested primer
And a second PCR performed with the T7 primer produced a second specific product, confirming the specificity of the primary PCR.

The PCR product was ligated into the plasmid pT7 Blue (Novagen) and the SEM protocol (Inoue, H. et al., Gene, 96:23 (1990)).
) Was transformed into Escherichia coli DH5a. Transformants were cultured in LB medium (1% Bac) supplemented with 100 μg / ml ampicillin.
to-Tryptone, 0.5% yeast extract and 1% NaCl). Subsequently, the plasmid was lysed by the alkaline lysis method (Sambrook, J. et al., Mo.
circular Cloning, CSH Press, 125 pages (1988).
).

Using this plasmid as a template, primer 1 or primer 2
A positive reaction was obtained by PCR using with T7 primers.
Partial nucleotide sequencing of the # SDH2-1 fragment further confirmed that the derived amino acid sequence downstream of the primer 1 binding site was consistent with the experimentally determined N-terminal amino acid sequence. However, # SDH2-1 contained only part of the first subunit gene.

Example 5: Using a 1.53 kb DNA fragment as a probe, S
Isolation of λ GEM5-1 clone containing DH subunit gene) # SDH2-1 isolated in Example 4 was replaced with DIG Labeling
and Detection Kit (The DIG System Use
r's Guide for Filter Hybridization. 6
-9, Boehringer Mannheim (1993)).

G. To construct a genomic DNA library of suboxydans KCTC 2111, the method described in Example 4 was followed. suboxydan
Genomic DNA isolated from s KCTC 2111 was partially digested with Sau3AI. The partially digested DNA was electrophoresed on a 0.8% agarose gel and the 15-23 kb size DNA was subjected to QLAEX II Gel.
Elution was performed using the Extraction Kit (QIAGEN). The eluted DNA was then ligated into the BamHI site of a λ GEM-11 vector (Promega). The ligation mixture was packed into a phage lambda particle using Packagene In
Packaging was performed using the Vitro Packaging System (Promega) according to the instruction manual. E. FIG. coli LE392 cells,
TB medium supplemented with 0.2% maltose and 10 mM MgSO ₄ (1% B
(acto-Tryptone and 0.5% NaCl) at 30 ° C. and stored at 4 ° C. when the OD ₆₀₀ reached 0.6. The packaging mixture was added to the cell suspension and the mixture was incubated at 37 ° C. for 30 minutes to infect. To this mixture, 3 ml of dissolved (45 ° C.) T
B top agar (0.8% Bact in T13 medium containing 10 mM MgSO ₄ )
o-Agar) was added, gently vortexed and immediately poured into LB plates. The plate was inverted and incubated overnight at 37 ° C.

The λ phage plaque was immobilized on a nylon membrane (Amersham),
Then, this membrane is mixed with a pre-hybridization solution (5 × SSC, 1%
(W / v) blocking reagent, 0.1% N-lauroyl sarcosine (laur
oylsarcosine), 0.02% SDS and 50% (v / v) formamide) at 42 ° C. for 3 hours.
aid). Next, the membrane was hybridized using a hybridization solution (a DIG-labeled # SDH2-1 probe diluted in a prehybridization solution) at 42 ° C. for 16 hours. Eleven of the 20,000 plaques of phage lambda were plaque hybridized with the DIG-labeled # SDH2-1 probe (The DH plaque).
DIG System User's Guide for Filter H
ybridization. Boehringer Mannheim (199
3)) gave a positive signal.

Λ DNA was isolated from a positive λ clone and Lambda DNA
Purification was performed using Purification Kit (Stratagene). The isolated lambda DNA was digested with BamH1 and subjected to 0.7% agarose gel electrophoresis. The DNA fragments separated on this gel were transferred to a nylon membrane and analyzed again by Southern hybridization using # SDH2-1 as a probe under the same conditions as above. A clone that gave a positive signal was selected and a 15 kb insert DNA was removed from this clone by Xho.
I and subsequently cloned into the XhoI site of pBluescript SK to obtain λ GEM 5-1. The λ GEM 5-1 clone was mapped by digestion with several different restriction enzymes. FIG. 6 shows a restriction map of λGEM 5-1.

Example 6: Determination of the nucleotide sequence of the 5.7 kb Pstl fragment in the λ GEM 5-1 clone The positive clone λ GEM 5-1 obtained in Example 5 is mapped with several different restriction enzymes. And analyzed by Southern hybridization as described in Example 5. The 5.7 kb PstI fragment that hybridized with # SDH2-1 was subcloned and the nucleotide sequence was determined. To simplify DNA sequencing, three overlapping subclones S1, S2 and S3 were constructed using the restriction enzyme sets KpnI-PstI, NotI-SacII and PstI-SacI, respectively, as shown in FIG. . A set of deletion clones was used as Exo III-Mungbean
Each subclone was prepared using the Deletion Kit (Stratagene). The nucleotide sequencing reaction was performed using the Dye Terminator.
Cycle Sequencing Ready Reaction Kit
(Applied Biosystems) and the sequence is automatically D
NA sequencer (Applied Biosystems, Model 373)
Determined in A).

FIG. 8 shows 4,83 in the 5.7 kb PstI fragment (SEQ ID NO: 7).
1 shows a 0 bp nucleotide sequence. The sequenced DNA contains two open reading frames (ORFs) of 2,265 nucleotides and 1,437 nucleotides. The first ORF encodes a first subunit. Before the first subunit gene, Shine-D located at 651 bp to 654 bp
There is an algarno sequence "AGGA". The signal sequence of 34 amino acids of the first subunit is located at 665 bp to 766 bp of SEQ ID NO: 7. The coding sequence for the mature portion of the first subunit protein is located between 767 bp and 2,929 bp of SEQ ID NO: 7, encoding a 720 amino acid polypeptide. The derived N-terminal amino acid sequence is completely identical to the 15 amino acid residues obtained by N-terminal amino sequence analysis.

The first ORF was followed by a second ORF, which was separated by a short intergenic region. The second ORF encodes a second subunit. Shine-Dalgarno sequence of the second subunit gene (A
GGA) is found at 2,950 bp to 2,953 bp of SEQ ID NO: 8, and this structural gene is located at 2,964 bp to 4,400 bp of SEQ ID NO: 8. The second subunit gene encodes a 478 amino acid polypeptide including a 36 amino acid signal sequence. The derived amino acid sequence of the mature polypeptide showed a perfect match with the experimentally obtained sequence for a sample treated with pyroglutamate aminopeptidase. Downstream of the stop codon of the second subunit gene, an inverted repeat is found.

[0133] The calculated molecular weights of the mature proteins of the first and second subunits are 79 kDa and 48 kDa, respectively, which are
This is in good agreement with the experimental values of 75 kDa and 50 kDa, respectively, as determined by DS-PAGE.

In addition, a characteristic PQQ binding sequence (a consensus sequence characteristically present at the amino and carboxy termini of PQQ-dependent dehydrogenase) was also found to be present in the first subunit gene. In Table 5, the characteristic PQQ binding sequence at the amino terminal portion is shown as SEQ ID NO: 17, and the characteristic sequence present at the carboxy terminal portion is shown as SEQ ID NO: 18, where X represents any amino acid. The characteristic sequence at the amino terminus occurs at positions 812 bp to 898 bp, and the characteristic sequence at the carboxy terminus is at positions 1,490 bp to 1,555 b
occurs at p. These data indicate that the first subunit is pyrroloquinoline quinone (
Provide further evidence that PQQ) is included as a cofactor.

[0135]

[Table 5] In addition, a single heme binding sequence is located at positions 2,6 in the first subunit gene.
It occurs between 12 bp and 2,626 bp (SEQ ID NO: 19 in Table 6 below), and it has also been found that three heme binding sequences occur at the following positions: 3,129 bp to 3, in the second subunit gene. 143 bp; 3,573 bp to 3
, 587 bp and 3,981 bp to 3,995 bp (where X _A represents any amino acid; X _B is any amino acid different from X _A ).

[0136]

[Table 6] A database search for homologous sequences was performed using the DNA of the first subunit gene.
The sequence was determined to show a large degree of similarity to many dehydrogenases containing pyrroloquinoline quinone (PQQ) as a cofactor. In particular, Acetob
alcohol dehydrogenase of actor polyoxygenes (Tam
aki, T .; Et al., Biochim. Biophys. Acta, 1088, 29
2 (1991)) and alcohol dehydrogenase from Acetobacter aceti (Inoue, T. et al., J Bacteriol, 171, 311).
5 (1989)) are 77% and 70% identical to the first subunit gene, respectively. Searching the database with the second subunit gene provides the greatest degree of similarity and suboxydans IFO 12528 cytochrome c (Takeda and Shimizu, J Ferm. Bioeng)
. 72: 1 (1991)) with 83% nucleotide sequence identity and 88% amino acid sequence identity.

Example 7: Design of primers and PCR cloning of a 320 bp DNA fragment containing part of the third subunit gene First subunit gene and second cloning described in Example 6 Nucleotide sequence analysis of the subunit gene indicated that the isolated operon clone did not contain a third subunit gene. Further sequencing of the 5 'and 3' flanking regions also failed to indicate the presence of the third subunit. Therefore, in order to isolate the gene encoding the third subunit, a degenerate primer for generating by PCR a short DNA fragment containing a part of the third subunit gene used as a probe, It was synthesized based on amino acid sequence information.

The N-terminal amino acid sequence of the mature third subunit protein obtained in Example 2 (SEQ ID NO: 11) and one of the internal amino acid sequences of the trypsinized peptide obtained in Example 3 ( Based on SEQ ID NO: 13), two degenerate primers, primer 3 (5'-GGGAATTC TTT (C)
CAA (G) GAA (G) ATG AAT (C) AA-3 ′) (SEQ ID NO: 20) and primer 4 (5′-GGGAATTC TT GAA A (G)
CC NGC A (G) TC A (G) TA-3 ′) (SEQ ID NO: 21) was synthesized.

The PCR reaction was performed with Takeda and Shimizu (Takeda and Sh
imizu, J .; Ferm. Bioeng. , 721 (1991)). genome DN of suboxydans KCTC 2111
A was performed with primers 3 and 4 using A as a template. This reaction produced a 320 bp DNA fragment. This 320 bp PCR product was ligated into pBluescript SK (Stratagene) and
EM protocol (Inoue, H. et al., Gene, 96, 23 (1990)). coli DH5α. 100 μg / m of transformant
Culture was performed in LB medium supplemented with 1 ampicillin. Subsequently, this plasmid was used in an alkaline lysis method (Sambrook, J. et al., Molecular Clon).
ing, CSH Press, (1988)). Subcloning was confirmed by performing a PCR reaction using this plasmid DNA as a template with primers 3 and 4. Partial nucleotide sequencing of the 320 bp fragment further confirmed that the derived amino acid sequence downstream of the primer 3 binding site was consistent with the experimentally determined N-terminal amino acid sequence. However, the 320 bp fragment contained only the N-terminal portion of the third subunit gene.

Example 8: Using a 320 bp DNA fragment as a probe,
Isolation of the subunit gene of Example 3) The 320 bp DNA fragment isolated in (Example 7) was
Labeling and Detection Kit (The DIG S
system User's Guide for Filter Hybrid
zation. 6-9, Boehringer Mannheim (1993)
)). Takeda and Shimizu (Takeda and Shmizu
imizu, J .; Ferm. Bioeng. G., 72, ((1991)). genome D isolated from suboxydans KCTC 2111
NA is digested with BamHI, ClaI, EcoRI, HindIII, PstI or XhoI, electrophoresed on a 0.8% agarose gel, and described on a nylon membrane (NYTRAN, Schleicher & Schuell) (Southern, EM. J. Mol. Biol. 98, 503 (1
975)). The membrane is placed in a hybridization oven (Hybaid) in a prehybridization solution (5 × SSC, 1% (
w / v) blocking reagent, 0.1% N-lauroyl sarcosine, 0.2%
(SDS and 50% (v / v) formamide) at 42 ° C. for 2 hours. Next, this membrane was added to a hybridization solution (
(DIG-labeled probe diluted in the pre-hybridization solution) at 42 ° C. for 12 hours. Southern hybridization is
A strong separate signal was given for each enzyme used. The DNA corresponding to the positive signal at 4.5 kb ClaI was eluted and pBluescript
A mini-library was constructed by cloning into ptSK. This mini-library was screened for positive clones by repeating Southern hybridization as described above. A clone giving a positive signal was selected and designated ClaI- # 69. FIG. 9 shows ClaI- # 6
9 represents the restriction map of Example 9.

Example 9 Nucleotide Sequence Analysis of a 4.5 kb ClaI Fragment Containing the Third Subunit The nucleotide sequence of the third subunit gene in the 4.5 kb ClaI- # 69 clone was determined, and analyzed. To facilitate DNA sequencing, several overlapping restriction fragments were subcloned and the nucleotide sequence of each clone was determined. Nucleotide sequencing reactions with Dye Ter
Minor Cycle Sequencing Ready React
ion Kit (Applied Biosystems), and the sequence was sequenced using an automated DNA sequencer (Applied Biosystems,
Model 373A).

FIG. 10 shows the 2700 bp of the 4.5 kb ClaI fragment (SEQ ID NO: 8).
1 shows the nucleotide sequence of The sequenced DNA contains a 921 nucleotide open reading frame (ORF), which encodes a third subunit polypeptide. Before the third subunit gene, 1,37
There is a potential Shine-Dalgarno sequence (AGG) located between 5 bp and 1,377 bp. The amino acid signal sequence of the third subunit polypeptide is located between 1,384 bp and 1,461 bp. The coding sequence for the mature part of the third subunit protein is located between 1,462 and 2,304 bp,
Encodes a 0 amino acid polypeptide. The derived N-terminal amino acid sequence is
Fully matched the 25 amino acid residues obtained by N-terminal amino acid sequence analysis. The calculated molecular weight of the mature protein of the third subunit was 29,552 Da, which was in good agreement with the experimental value of 29 kDa determined by SDS-PAGE.

[0143]

[Table 7]

[0144]

[Table 8]

[0145]

[Table 9]

[0146]

[Table 10]

[Brief description of the drawings]

FIG. 1 depicts DEAE-TSK column chromatography using pH 5.0 (A) and pH 6.0 (B) sodium acetate buffers.

FIG. 2 shows SDS-PAGE analysis of peak I fraction (A) and peak II fraction (B) separated by DEAE-TSK column chromatography.

FIG. 3 shows DEAE-TSK column chromatography using a pH 6.5 sodium phosphate buffer.

FIG. 4 is an SDS-PAGE analysis of peak I (lane 1), peak II (lane 3) and peak III (lane 2) column fractions separated by DEAE-TSK column chromatography at pH 6.5. Represents Lane M shows the molecular weight standard marker.

FIG. 5 shows an HPLC chromatogram of a tryptic digest of peak II protein from DEAE-TSK column chromatography at pH 6.5. The dotted line indicates the concentration gradient of acetonitrile in the mobile phase.

FIG. 6 shows a restriction map of λGEM 5-1. 1. Used as probe
The 53 kb DNA fragment (# SDH2-1) is shown as a black bar.

FIG. 7: S1 DNA fragment, S2 DN, generated from a 5.7 kb PstI fragment of λ GEM 5-1 using different sets of restriction enzymes.
Represents the location of the A and S3 DNA fragments.

FIG. 8 shows 4,830 bases of a 5.7 kb PstI fragment (SEQ ID NO: 7).
Represents the nucleotide sequence of The deduced amino acid sequences for the first and second subunits are shown below the nucleotide sequences. The N-terminal amino acid sequence obtained by N-terminal amino acid sequencing of purified sorbitol dehydrogenase is underlined. The signal sequence cleavage site is shown as a triangle. In the heme binding sequence,
Underlined with dotted line. Potential ribosome binding sequences (SD) are boxed. The transcription termination stem-loop structure is indicated by an arrow. The complete coding sequence for the first subunit gene is located at positions 665-2,929 (SEQ ID NO: 1), the signal sequence is located at positions 665-766, and the first subunit of SDH The coding sequence for the mature protein is located between positions 767 and 2,929 (SEQ ID NO: 22). The complete coding sequence for the second subunit gene is 2,96
Located at positions 4 to 4,400 (SEQ ID NO: 2), the signal sequence is from 2,964 to 3
, 071 and the coding sequence for the mature protein of the second subunit of SDH is located at positions 3,072-4,400 (SEQ ID NO: 23).

FIG. 9 shows a restriction map of ClaI- # 69. The closed square represents the coding region of the third subunit gene of sorbitol dehydrogenase.

FIG. 10 shows the nucleotide sequence of the DNA fragment containing the third subunit gene of sorbitol dehydrogenase (SEQ ID NO: 8). The deduced amino acid sequence is shown below this nucleotide sequence. The signal sequence cleavage site is shown as a triangle. Potential ribosome binding sequences (SD) are boxed. The complete coding sequence for the third subunit gene is located at positions 1,384-2,304 (SEQ ID NO: 3), the signal sequence is located at positions 1,384-1,461 and the SDH The coding sequence for the mature protein of the third subunit is from position 1,462 to 2
, 304 (SEQ ID NO: 24).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 9/02 C12P 21/02 C C12P 19/02 C12N 15/00 ZNA 21/02 5/00 A (81 ) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, N, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ , LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZA, ZW (71) Applicant Aker-Daniels-Midland Company United States Illinois 62525, Dicatar, Box 1470, Ferries Parkway 4666 (72) Inventor Cho, Yi-sun Republic of Korea 305-600 Daejeon, Yousung-k, Khun-dong, Tasol Apartments 395-3 102-507 (72) Inventor A, San-ki South Korea 143-210 Seoul, Kwangjin-k, Kwangjang-dong, Cardon 101, Kuk-dong villa (72) Inventors A, Une Republic of Korea 302-230 Daejeon, Soak, Joungnim-dong, Usun apartment 112- 1305 F-term (Reference) 4B024 AA01 AA05 BA08 CA02 CA09 DA05 GA11 HA03 4B050 CC03 DD02 LL01 LL02 4B064 AF02 CA02 CA19 CC24 DA01 4B065 AA28 AB01 BA02 CA23 CA28 CA42 CA44

Claims (34)

[Claims] 1. An isolated nucleic acid molecule comprising: (a) a polynucleotide of SEQ ID NO: 1; (b) a polynucleotide fragment of at least about 20 nucleotides in length of the polynucleotide of SEQ ID NO: 1; A nucleic acid comprising a polynucleotide sequence selected from the group consisting of: a polynucleotide encoding the amino acid sequence of SEQ ID NO: 4; and (d) a polynucleotide encoding a fragment of at least about 10 amino acids in length of the amino acid sequence of SEQ ID NO: 4. molecule. 2. An isolated nucleic acid molecule comprising a polynucleotide that is at least about 95% identical to the isolated nucleic acid molecule of claim 1. 3. A vector comprising the nucleic acid according to claim 1. 4. A process for producing a vector according to claim 3, comprising: (a) inserting the polynucleotide according to claim 1 into the vector; and (b) A) selecting and propagating the vector in a host cell. 5. A host cell comprising the vector according to claim 3. 6. An isolated nucleic acid molecule comprising: (a) a polynucleotide of SEQ ID NO: 2 or a fragment thereof; (b) a polynucleotide fragment of at least about 20 nucleotides in length of the polynucleotide of SEQ ID NO: 2; (C) a polynucleotide encoding the amino acid sequence of SEQ ID NO: 5; and (d) a polynucleotide encoding a fragment of at least about 10 amino acids in length of the amino acid sequence of SEQ ID NO: 5. A nucleic acid molecule. 7. An isolated nucleic acid molecule comprising a polynucleotide that is at least about 95% identical to the isolated nucleic acid molecule of claim 6. 8. A vector comprising the nucleic acid according to claim 6. 9. A process for producing a vector according to claim 8, comprising: (a) inserting the polynucleotide according to claim 6 into the vector; and (b) A) selecting and propagating the vector in a host cell. 10. A host cell comprising the vector according to claim 8. 11. An isolated nucleic acid molecule comprising: (a) a polynucleotide of SEQ ID NO: 3; (b) a polynucleotide fragment of at least about 20 nucleotides in length of the polynucleotide of SEQ ID NO: 3; A nucleic acid comprising a polynucleotide sequence selected from the group consisting of: a polynucleotide encoding the amino acid sequence of SEQ ID NO: 6; and (d) a polynucleotide encoding a fragment of at least about 10 amino acids in length of the amino acid sequence of SEQ ID NO: 6. molecule. 12. An isolated nucleic acid molecule comprising a polynucleotide at least about 95% identical to the isolated nucleic acid molecule of claim 11. A vector comprising the nucleic acid according to claim 11. 14. A process for producing a vector according to claim 13, comprising: (a) inserting the polynucleotide according to claim 11 into the vector; and (b) A) selecting and propagating the vector in a host cell. 15. A host cell comprising the vector according to claim 13. 16. An isolated nucleic acid molecule comprising: (a) a polynucleotide of SEQ ID NO: 7; and (b) a polynucleotide fragment of at least about 20 nucleotides in length of the polynucleotide of SEQ ID NO: 7. A nucleic acid molecule comprising a polynucleotide sequence selected from the group. 17. An isolated nucleic acid molecule comprising a polynucleotide at least about 95% identical to the isolated nucleic acid molecule of claim 16. 18. A vector comprising the nucleic acid according to claim 16. 19. A process for producing a vector according to claim 18, comprising: (a) inserting the polynucleotide according to claim 16 into the vector; and (b) A) selecting and propagating the vector in a host cell. 20. A host cell comprising the vector according to claim 18. 21. The polynucleotide according to claim 12, wherein the polynucleotide is KCTC accession number 0593BP.
17. The nucleic acid molecule of claim 16, having the complete nucleotide sequence of a DNA clone contained in. 22. An isolated nucleic acid molecule, comprising: (a) a polynucleotide of SEQ ID NO: 8; and (b) a polynucleotide fragment of at least about 20 nucleotides in length of the polynucleotide of SEQ ID NO: 8. A nucleic acid molecule comprising a polynucleotide sequence selected from the group. 23. An isolated nucleic acid molecule comprising a polynucleotide at least about 95% identical to the isolated nucleic acid molecule of claim 22. 24. A vector comprising the nucleic acid according to claim 22. 25. A process for producing a vector according to claim 24, comprising: (a) inserting the polynucleotide according to claim 22 into the vector; and (b) A) selecting and propagating the vector in a host cell. 26. A host cell comprising the vector according to claim 24. 27. The polynucleotide, wherein the polynucleotide is KCTC accession number 0594BP.
23. The nucleic acid molecule of claim 22, having the complete nucleotide sequence of a DNA clone contained in. 28. A process for producing L-sorbose from D-sorbitol, comprising: (a) the following: (b) a polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 1; (c) SEQ ID NO: 2 Transforming a host cell with at least one isolated nucleotide sequence selected from the group consisting of: a polynucleotide comprising the polynucleotide sequence of: and (d) a polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 3; And (e) selecting and growing the transformed host cell. 29. The process of claim 28, wherein said host cell is a Gluconobacter. 30. An isolated polypeptide, comprising: (a) a polypeptide sequence encoded by the polynucleotide sequence of SEQ ID NO: 3; (b) a polypeptide sequence of SEQ ID NO: 6; and (c) A polypeptide having a length of at least about 10 amino acids derived from the polypeptide sequence of SEQ ID NO: 6. A polypeptide comprising a polypeptide sequence selected from the group consisting of: 31. A process for producing a polypeptide, comprising: (a) growing the host cell of claim 15; and (b) expressing the polypeptide of claim 29. And c) isolating the polypeptide. 32. A process for increasing the production of 2-keto-L-gulonic acid, comprising: (a) the following: (b) a polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 1; (c) Transforming a host cell with at least one isolated nucleotide sequence selected from the group consisting of: a polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 2; and (d) a polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 3. And e) selecting and growing said transformed host cells. 33. An isolated nucleic acid molecule, comprising: (a) nucleotides 1 to 664 of SEQ ID NO: 7, identified as SEQ ID NO: 28.
(B) nucleotides 50-66 of SEQ ID NO: 7, identified as SEQ ID NO: 29
4; (c) nucleotides 100-6 of SEQ ID NO: 7, identified as SEQ ID NO: 30
64; (d) nucleotides 150-6 of SEQ ID NO: 7, identified as SEQ ID NO: 31
64; (e) nucleotides 200-6 of SEQ ID NO: 7, identified as SEQ ID NO: 32.
64; (f) nucleotides 250-6 of SEQ ID NO: 7, identified as SEQ ID NO: 33
64; (g) nucleotides 300-6 of SEQ ID NO: 7, identified as SEQ ID NO: 34.
64; (h) nucleotides 350-6 of SEQ ID NO: 7, identified as SEQ ID NO: 35
64; (i) nucleotides 400-6 of SEQ ID NO: 7, identified as SEQ ID NO: 36
64; (j) nucleotides 450-6 of SEQ ID NO: 7, identified as SEQ ID NO: 37
64; (k) nucleotides 500-6 of SEQ ID NO: 7, identified as SEQ ID NO: 38
64; (l) nucleotides 550-6 of SEQ ID NO: 7, identified as SEQ ID NO: 39
64; (m) nucleotides 600-6 of SEQ ID NO: 7, identified as SEQ ID NO: 40.
64; (n) nucleotides 665-2, SEQ ID NO: 7, identified as SEQ ID NO: 1.
929, a nucleotide sequence encoding the full length subunit 1 protein of the SDH of the invention; (o) nucleotides 767-2 of SEQ ID NO: 7, identified as SEQ ID NO: 22
929, a nucleotide sequence encoding the subunit 1 protein of the mature form of SDH of the invention; (p) nucleotides 2,930 of SEQ ID NO: 7, identified as SEQ ID NO: 41.
(Q) nucleotides 2,964 to SEQ ID NO: 7, identified as SEQ ID NO: 2;
4,400 nucleotide sequences encoding the SDH full length subunit 2 protein of the invention; (r) nucleotides 3,072 of SEQ ID NO: 7, identified as SEQ ID NO: 23
-4,400 nucleotide sequences encoding the subunit 2 protein of the mature form of SDH of the invention; (s) nucleotides 4,401 of SEQ ID NO: 7, identified as SEQ ID NO: 42.
-4,451; (t) nucleotides 4,401 of SEQ ID NO: 7, identified as SEQ ID NO: 43
-4,501; (u) nucleotides 4,401 of SEQ ID NO: 7, identified as SEQ ID NO: 44
-4,551; (v) nucleotides 4,401 of SEQ ID NO: 7, identified as SEQ ID NO: 45
-4,601; (w) nucleotides 4,401 of SEQ ID NO: 7, identified as SEQ ID NO: 46
-4,651; (x) nucleotides 4,401 of SEQ ID NO: 7, identified as SEQ ID NO: 47
-4,701; (y) nucleotides 4,401 of SEQ ID NO: 7, identified as SEQ ID NO: 48
-4,751; (z) nucleotides 4,401 of SEQ ID NO: 7, identified as SEQ ID NO: 49
-4,801; and (aa) nucleotides 4,40 of SEQ ID NO: 7, identified as SEQ ID NO: 50
A nucleic acid molecule comprising a polynucleotide sequence selected from the group consisting of: 34. An isolated nucleic acid molecule, comprising: (a) nucleotides 1-1,3 of SEQ ID NO: 8, identified as SEQ ID NO: 51.
83; (b) nucleotides 50-1 of SEQ ID NO: 8, identified as SEQ ID NO: 52;
383; (c) nucleotides 100-1 of SEQ ID NO: 8, identified as SEQ ID NO: 53
383; (d) nucleotides 150-1 of SEQ ID NO: 8, identified as SEQ ID NO: 54.
383; (e) nucleotides 200-1 of SEQ ID NO: 8, identified as SEQ ID NO: 55.
383; (f) nucleotides 250-1 of SEQ ID NO: 8, identified as SEQ ID NO: 56.
383; (g) nucleotides 300-1 of SEQ ID NO: 8, identified as SEQ ID NO: 57.
383; (h) nucleotides 350-1 of SEQ ID NO: 8, identified as SEQ ID NO: 58
383: (i) nucleotides 400-1 of SEQ ID NO: 8, identified as SEQ ID NO: 59
383; (j) nucleotides 450-1 of SEQ ID NO: 8, identified as SEQ ID NO: 60.
383; (k) nucleotides 500-1 of SEQ ID NO: 8, identified as SEQ ID NO: 61.
383; (l) nucleotides 550-1 of SEQ ID NO: 8, identified as SEQ ID NO: 62.
383; (m) nucleotides 600-1 of SEQ ID NO: 8, identified as SEQ ID NO: 63.
383; (n) nucleotides 600-1 of SEQ ID NO: 8, identified as SEQ ID NO: 64.
383; (o) nucleotides 650-1 of SEQ ID NO: 8, identified as SEQ ID NO: 65
383; (p) nucleotides 700-1 of SEQ ID NO: 8, identified as SEQ ID NO: 66.
383; (q) nucleotides 750-1 of SEQ ID NO: 8, identified as SEQ ID NO: 67.
383; (r) nucleotides 800-1 of SEQ ID NO: 8, identified as SEQ ID NO: 68.
383; (s) nucleotides 850-1 of SEQ ID NO: 8, identified as SEQ ID NO: 69.
383; (t) nucleotides 900-1 of SEQ ID NO: 8, identified as SEQ ID NO: 70.
383: (u) nucleotides 950-1 of SEQ ID NO: 8, identified as SEQ ID NO: 71
(V) nucleotides 1,000 of SEQ ID NO: 8, identified as SEQ ID NO: 72.
(W) nucleotides 1,050 of SEQ ID NO: 8, identified as SEQ ID NO: 73
(X) nucleotides 1,100 of SEQ ID NO: 8, identified as SEQ ID NO: 74
(Y) nucleotides 1,150 of SEQ ID NO: 8, identified as SEQ ID NO: 75
(Z) nucleotides 1,200 of SEQ ID NO: 8, identified as SEQ ID NO: 76
(Aa) nucleotides 1,25 of SEQ ID NO: 8, identified as SEQ ID NO: 77
(Bb) nucleotides 1,30 of SEQ ID NO: 8, identified as SEQ ID NO: 78
(Cc) nucleotides 1,35 of SEQ ID NO: 8, identified as SEQ ID NO: 79
(Dd) nucleotides 1,384 of SEQ ID NO: 8, identified as SEQ ID NO: 3
Nucleotide sequences encoding the full-length SDH subunit 3 protein of the present invention from ｅ1,461; (ee) nucleotides 1,46 of SEQ ID NO: 8, identified as SEQ ID NO: 24
2-2,304 nucleotide sequences encoding mature forms of the SDH subunit 3 protein of the invention; (ff) nucleotides 2,30 of SEQ ID NO: 8, identified as SEQ ID NO: 80
(Gg) nucleotides 2,30 of SEQ ID NO: 8, identified as SEQ ID NO: 81
(Hh) nucleotides 2,30 of SEQ ID NO: 8, identified as SEQ ID NO: 82
(Ii) nucleotides 2,30 of SEQ ID NO: 8, identified as SEQ ID NO: 82
(Jj) nucleotides 2,30 of SEQ ID NO: 8, identified as SEQ ID NO: 83
(Kk) nucleotides 2,30 of SEQ ID NO: 8, identified as SEQ ID NO: 85
(11) nucleotides 2,30 of SEQ ID NO: 8, identified as SEQ ID NO: 86
5-2,655; and (mm) nucleotides 2,30 of SEQ ID NO: 8, identified as SEQ ID NO: 87.
5-2,700; a nucleic acid molecule comprising a polynucleotide sequence selected from the group consisting of: